The alpha(2) subunit of the VLA-2 receptor (CD49B) was mapped to human chromosome 5 by several independent approaches. First, the expression of the alpha(2) subunit at the protein level was investigated in a panel of human-mouse hybrid cell lines. Cell surface expression was detected by indirect immunofluorescence with monoclonal anti-alpha(2) antibody 12F1. Intracellular alpha(2) antigen was detected by immunostaining of whole cell extracts or of immunoprecipitated 12F1 antigen with the monoclonal antibodies 3H8 and 5C5. Second, the presence of human genomic alpha(2) sequences in the panel of human-mouse hybrids was detected by PCR, using primers derived from the published alpha(2) cDNA sequence. The specificity of the amplification product was shown by direct sequencing. The results of the PCR study were confirmed by amplifying a CD14 gene fragment, known to map to chromosome 5. Finally, in situ hybridization with a H-3-labeled 1040-bp cDNA probe, also obtained by PCR, confirmed and refined the localization of CD49B on chromosome 5 at q23-31.